Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices.

The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosine A(2b) receptors on astrocytes induces increased synthesis and release of interleukin-6, which also exerts neuroprotective effects, we hypothesized that the effects of interleukin-6 and of adenosine might be related. We report here that stimulation with interleukin-6 of cultured astrocytes, of cultured organotypic brain slices from newborn rat cortex, and of freshly prepared brain slices from rat cortex induces a concentration- and time-dependent upregulation of adenosine A(1) receptor mRNA. This increased adenosine A(1) receptor mRNA expression is accompanied in astrocytes by an increase in adenosine A(1) receptor-mediated signaling via the phosphoinositide-dependent pathway. Since upregulation of adenosine A(1) receptors leads to increased neuroprotective effects of adenosine, we suggest that the neuroprotective actions of interleukin-6 and adenosine are related and might be mediated at least in part through upregulation of adenosine A(1) receptors. These results may be of relevance for a better understanding of neuroprotection in brain damage but also point to a potential impact of neuroprotection in the mechanisms of the antidepressive effects of chronic carbamazepine, electroconvulsive therapy, and sleep deprivation, which are all accompanied by adenosine A(1) receptor upregulation.

Inhibition of inositol uptake in astrocytes by antisense oligonucleotides delivered by pH-sensitive liposomes.

An oligonucleotide of 20 bases, complementary to a region of the sodium/myo-inositol cotransporter (SMIT) mRNA, was used to investigate the uptake efficiency and activity of transferred antisense oligonucleotides with regard to substrate uptake. We compared the efficiency of oligonucleotide delivery after application of either free or liposome-encapsulated material. Delivery of liposome-encapsulated material (marker or oligonucleotides) into astrocytoma cells and primary astrocyte cultures was more effective with pH-sensitive liposomes [dioleoylphosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS)] than with non-pH-sensitive liposomes (soy lecithin) or free material in solution. Antisense activity was evaluated by determination of myo-inositol uptake and detection of SMIT transcripts by RT-PCR. Encapsulation of oligonucleotides in pH-sensitive liposomes increased the inhibition of inositol uptake at least 50-fold compared with application of free oligonucleotides in solution.

Differential expression, activity and regulation of the sodium/myo-inositol cotransporter in astrocyte cultures from different regions of the rat brain.

The high-affinity sodium/myo-inositol cotransporter (SMIT) is involved in osmoregulation in several cells and tissues. In the CNS the activity of SMIT also determines the individual susceptibility of neural cells to the inositol depleting effect of lithium, which is considered to be important in lithium's therapeutic effects in manic-depressive illness. Among neural cells SMIT is particularly active in astrocytes. In the present work we have cloned the cDNA of SMIT of the rat and assessed its activity, expression and regulation in primary astroglia cultures derived from five different rat brain regions: cerebellum, cortex, diencephalon, hippocampus and tegmentum. After an incubation period of 24 h in medium containing 3[H]labeled myo-inositol different steady-state concentrations were detected which were dependent on the brain region from which the astrocytes were cultured. In addition, myo-inositol uptake in astrocytes from different areas was characterized by two different Km values (27 microM for cerebellum and diencephalon, 50 microM for cortex, hippocampus and tegmentum) and by three different v(max) values (approx. 200 pmol/mg protein/min for astrocytes from cerebellum and tegmentum, 298 for hippocampus and 465 for cortex), indicating that the active myo-inositol uptake into astroglial cells is distinct in the various brain regions. The efficacy of uptake as determined by v(max) values of 3[H]myo-inositol uptake correlated with the level of mRNA of SMIT in the astrocyte cultures from the various brain regions as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Both 3[H]myo-inositol uptake and SMIT mRNA content was upregulated by incubation of astrocytes in medium of increased osmolarity. In astrocytes from cerebellum, cortex, hippocampus and tegmentum 3[H]myo-inositol uptake was downregulated by chronic incubation with 400 microM inositol. This effect was not observed in astrocytes from diencephalon. Furthermore, in astrocytes from cortex and hippocampus but not from cerebellum, diencephalon and tegmentum incubation with corticosterone for three days upregulated 3[H]myo-inositol uptake. It is concluded that SMIT is differentially expressed and regulated in astrocytes from distinct brain regions. These regional differences suggest particular consideration of localized effects in investigations of the role of myo-inositol in the mechanism of action of antibipolar drugs.

Inhibition of the high affinity myo-inositol transport system: a common mechanism of action of antibipolar drugs?

The mechanism of action of antibipolar drugs like lithium, carbamazepine, and valproate that are used in the treatment of manic-depressive illness, is unknown. Lithium is believed to act through uncompetitive inhibition of inositolmonophosphatase, which results in a depletion of neural cells of inositol and a concomitant modulation of phosphoinositol signaling. Here, we show that lithium ions, carbamazepine, and valproate, but not the tricyclic antidepressant amitriptyline, inhibit at therapeutically relevant concentrations and with a time course similar to their clinical actions the high affinity myo-inositol transport in astrocyte-like cells and downregulate the level of the respective mRNA. Inhibition of inositol uptake could thus represent an additional pathway for inositol depletion, which might be relevant in the mechanism of action of all three antibipolar drugs.

Differential expression of inflammatory mediators in rat microglia cultured from different brain regions.

Microglial cells show a rather uniform distribution of cell numbers throughout the brain with only minor prevalences in some brain regions. Their in situ morphologies, however, may vary markedly from elongated forms observed in apposition with neuronal fibers to spherical cell bodies with sometimes extremely elaborated branching. This heterogeneity gave rise to the hypothesis that these cells are differentially conditioned by their microenvironment and, therefore, also display specific patterns of differential gene expression. In this study, microglia were isolated from 2-4 week-old mixed CNS cultures that had been prepared from neonatal rat diencephalon, tegmentum, hippocampus, cerebellum and cerebral cortex, and were investigated 24 h later. Messenger RNA levels of proteins involved in crucial immune functions of this cell type (TNF-alpha, CD4, Fcgamma receptor II, and IL-3 receptor beta-subunit) have been determined by semi-quantitative RT-PCR. The results clearly show, that three of these mRNAs (TNF-alpha, CD4, Fcgamma receptor II) are differentially expressed in microglia with hippocampal microglia displaying the highest levels of these mRNAs. The data strongly support the notion that the status of microglial gene expression depends on their localization in brain and on specific interactions with other neural cell types. Consequently, it is hypothesized that their responsiveness to signals arising in injury or disease may vary from one brain region to another.

Lithium-induced inositol depletion in rat brain after chronic treatment is restricted to the hypothalamus.

The influence of acute and chronic lithium administration on inositol content was examined in five different regions of the rat brain: caudate, cerebellum, cortex, hippocampus and hypothalamus. After acute administration of lithium at doses of 3, 6 or 10 mEq kg-1, no significant reductions of inositol were found in any brain region. Also no significant changes were observed in cortex, caudate, hippocampus and cerebellum after chronic treatment with lithium-containing diet, which led to brain concentrations of lithium in the therapeutic range. However, a moderate but significant reduction of inositol was under these conditions observed in the hypothalamus. At basal conditions, ie in control rats not treated with lithium, the inositol content in various brain areas was different, the hypothalamus containing the highest inositol concentration (4.4 mmol kg-1 wet weight) and the cortex the lowest (2.3 mmol kg-1 wet weight). It is concluded that chronic lithium treatment at therapeutically relevant brain concentrations does not evoke major changes in the inositol content of the brain but induces a moderate decrease which is restricted to the hypothalamus. The results are discussed with respect to the potential function of the hypothalamus in affective disorders.

Differential uptake of myo-inositol in vivo into rat brain areas.

Oral inositol has been reported to have antidepressant and antipanic properties in humans. Inositol enters the brain poorly and high doses are required. Natural uptake processes and specific transporters are involved. We here report that intraperitoneally administered inositol is taken up differently by various brain areas and that brain areas have different baseline inositol levels. These effects could be important in understanding the differential effects of lithium-induced lowering of inositol and of behavioral effects of exogenous inositol.